ISSN (Online) 2348 – 7968 | Impact Factor (2016) – 5...

IJISET - International Journal of Innovative Science, Engineering & Technology, Vol. 4 Issue 12, December 2017

ISSN (Online) 2348 – 7968 | Impact Factor (2016) – 5.264

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Comprehensive Management for Wilt Disease Caused By Fusarium Oxysporum In

Tomato Plant Mohamed S. Attia, Ahmed M. Younis, Ayman F. Ahmed and Amer M. Abd Elaziz

Botany and Microbiology Department, Faculty of Science, Al-Azhar University,11884 Nasr City, Cairo, Egypt.

*[email protected]

Abstract The plant growth promoting from different sources were tested to enhance plant

growth and suppress plant diseases in tomato plant, these include rhizobacteria (Bacillus subtilis, Serratia marcescens), Cyanobacteria (Nostoc muscorum, Anabaena oryzae), a plant water extract (Salix, Artemisia), antagonistic fungal species (Trichoderma (T) harzianum and Ganoderma (G) lucidum). The current study was carried out at, experimental farm station of Botany and Microbiology Department, Faculty of Science, Al-Azhar University; to investigate the efficient antagonistic these inducers against Fusarium wilt disease in tomato plant under filed experiment. Disease symptoms, disease index, phytochemicals and antifungal protein as well as isozyme markers as response to induction SR in tomato plants were recorded. The results demonstrated that F. oxysporum f. sp Lycopersici challenged plants treated with T.harzianum as well as G. lucidum extracts which showed the highest significant reduction in percent disease infection (PDI) with 4.16%, followed by treatment with A. oryzae as well as Artemisia water extract showed (8.33%), then S. marcescens as well as N. muscorum extracts showed (25%). Also, Salix water extract as well as B. subtilis with (33.33%) compared with control infected plants (83.33%). Considerable increase in all tested phytochemical parameters of tomato plants were obtained due to use of the tested elicitors than control infected plants. The beneficial effects of the tested inducers were extended to increase not only salicylic acid (SA), Abscisic acid (ABA), Indole acetic acid (IAA) and Gibberellin (GA3), but also the activities of peroxidase and polyphenol oxidase enzymes in comparison with control. a new pattern of pathogenesis related proteins (PRS) were produced, also the results appeared that tomato plants treated with inducers show variability in number, relative mobility and density of polypeptide bands of peroxidase and polyphenol oxidase isozymes according to the type of elicitors used. Key words: Tomato plant – Fusarium oxysporum - Plant growth promoting rhizobacteria – Cyanobacteria - Ganoderma lucidum - iso-zymes – Biotic and abiotic. Introduction:

Fusarium wilt of tomato considered one of the most serious diseases of tomato in

field as well as greenhouse-grown tomatoes worldwide (Amini and Sidovich, 2010). The

fungus can be found as soil borne, air borne or on plant residue and can be transmitted

through any part of the plant (Summeral et al., 2003). The wilt caused by F. oxysporum is

appear as wilt plants, yellowed leaves and significantly decreasing the quantity and the

quality of the crop (Ajigbola and Babalola, 2013 and Akram et al., 2013). Pathogenic

problems can be decreased or elimination through exogenous application of biotic or abiotic

elicitors that induce resistance which can be categorized either as systemic acquired (SAR)

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or induced systemic resistance (ISR). Induced resistance (SAR and ISR) involves the

synchronized action of multiple genes and/or defense signaling pathways. Although the

downstream components are similar in SAR and ISR mechanisms, upstream components

differ, mainly involving the salicylic acid and jasmonic acid/ethylene pathways for SAR and

ISR induction, respectively (Pieterse and Van Loon, 2007). A number of plant species have

been reported to possess natural substances that are toxic to several plant pathogenic fungi

(Goussous et al. 2010). In addition, Sivaprakasam et al. (2011) find that aqueous extract of

fruiting bodies from G. lucidum show strong antifungal activity against fungal pathogens

including F. oxysporum. Plant growth promoting rhizo-bacteria (PGPR) have various

mechanisms one of which is regulating hormonal and nutritional balance, inducing resistance

against plant pathogens, and solubilizing nutrients for easy uptake by plants. In addition,

PGPR show synergistic and antagonistic interactions with microorganisms within the

rhizosphere which indirectly enhance the growth of plant (Vejan et al., 2016). Also, may be

producing various active compounds which induced resistance against plant pathogens

(Ragaa and Mostafa,2013). Cyanobacteria have also been studied for the control of plant

pathogenic fungi, particularly soil borne diseases (Tassara et al., 2008). Dipotassium

hydrogen phosphate application in plants for induction of resistance is based on the activity

of enzymes related to the cell wall structure (Olivieri et al 2012).

2 Materials and methods

Plant material: For the present study, well identified Four Weeks-Tomato seedlings (Solanum

Lycopersicon L. cv. Castle rock II PVP) were kindly obtained from agricultural research

center (ARC), ministry of agriculture, Giza, Egypt.

Isolation and maintenance of F. oxysporum:

Fusarium oxysporum was isolated from symptomatic diseased tomato plants

according to (Katan et al., 1991), and identified morphologically macroscopic and

microscopic according to (Nelson et al., 1983 and Leslie and Sumerell, 2006). then

pathogen was confirmed by pathogenicity test according to (Hibar et al., 2007).

Maintenance of stock culture, Fusarium oxysporum was preserved on slants at 0oc on

molt extract agar medium (MEA).

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Source and application methods of elicitors:

Tow bacterial strains Bacillus subtilis, and Serratia marcescens were obtained kindly

from Bio-log Technique at Bio-fertilizer production unit, Soil, Water and Environment

Research institute, Agricultural Research centre (ARC), Giza, Egypt. The inoculum

suspensions were approximately adjusted to 109 CFU/ml culture (colony forming unit). Tow

strains of Cyanobacteria (Nostoc muscorum, Anabaena. oryzae) were obtained kindly from

the Microbiology Department; Soils, Water and Environment Research Institute (SWERI),

Agricultural Research, Centre (ARC). The inoculum suspensions were approximately

adjusted to 106 CFU/ml culture. The plant water extract of Salix, Artemisia obtained

According to (Adebolu and Oladimeji, 2007) dried leaves of both plants obtained from

Agriculture Research Centre (ARC) Giza Egypt. Trichoderma harzianum from the

Microbiology Department; Soils, Water and Environment Research Institute (SWERI),

Agricultural Research, Centre (ARC). Also, the fruiting bodies of Ganoderma lucidum was

obtained kindly from Dr. Younis and extracted according to the method described by Younis

Younis et al., 2014. Finally salicylic acid 10 mM (sigma company) and Di-potassium

hydrogen phosphate (K2HPO4) (sigma company) at concentration 100Mm were prepared.

Greenhouse experiment:

Tested elicitors were applied 7 days before inoculation with F. oxysporum. The

experiment was done in the garden of Faculty of Science, Al Azhar University Nasr city

Egypt in April 2017. Complete block design was used with ten treatments and two controls

(each has eight replicates). Treatments include; 1) healthy control (no fungus); 2) tomato

seedlings inoculated with F. oxysporum that served as infected control; 3) B. subtilis + F.

oxysporum; 4) S. marcescens + F. oxysporum; 5) N. muscorum + F. oxysporum; 6) A.

oryzae + F. oxysporum; 7)Artemisia extract + F. oxysporum; 8)Salix extract + F.

oxysporum; 9) salicylic acid + F. oxysporum; 10) Dipotassium hydrogen phosphate + F.

oxysporum; 11) T. harzianum + F. oxysporum; 12) G. leucidum + F. oxysporum .

Treatments were kept in the greenhouse under room temperature receiving water as required.

Tomato plants symptoms were followed and stage one determined after 30 days after F.

oxysporum inoculation and second stage was determined after 50 days.The plant samples

were collected carefully for studying metabolic and biochemical indicators of resistance in

tomato.

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Disease symptoms and disease index:

Disease symptoms were assessed after 60 days inoculation and the disease index was

evaluated according to (leath et al., 1989) with slight modifications using score consisting of

five classes: 0: (no symptoms), 1: (slight yellow of lower leaves), 2: (moderate yellow plant

), 3: (wilted plant), 4: (plants severely stunted and destroyed). Disease index (DI) was

calculated using the five-grade scale according to the formula: DI= (1n1+ 2n2 + 3n3 +

4n4)100/4nt. Where n1-n4 the number of plants in the indicated classes, and Nt total number

of plants tested. Protection % = A–B/A × 100% Where, A = PDI in infected control plants B

= PDI in infected- treated plants.

5-Metabolic and biochemical indicators of resistance in tomato: Determination of total soluble proteins (mg/100g of dry wt) according to the method

of (Lowery et al., 1951) using casein as a standard protein. Total soluble carbohydrate was

extracted according to (Said et al., 1964) and determined using anthrone technique according

to (Umbriet et al., 1969). Determination of phenolic compounds (mg/100g of dry wt) was

carried out according to that method described by (Daniel and George, 1972). Enzymes

extracted according to (MuKherjee and Choudhuri, 1983). SOD activity was determined

by measuring the inhibition of the auto-oxidation of pyrogallol using a method described by

(Marklund and Marklund, 1974). Peroxidase activity enzyme was determined according to

the method adopted by (Srivastava, 1987). The activity of polyphenol oxidase enzyme was

determined according to the method adopted by (Matta and Dimond, 1963). Determination

of endogenous hormones (IAA, GA and ABA) in the terminal buds of the treated plants as

well as the control were carried out as described by (Knegt and Brunima, 1973) Protein

finger print was analyzed using Sodium dodecyl sulfate -polyacrylamide gel electrophoresis

(SDS - PAGE) according to (Studier, 1973). Native-polyacrylamide gel electrophoresis

(Native-PAGE) was conducted to identify isozyme variations among the studied plants using

two isozyme systems. Peroxidase (Px) isozyme was determined according to the method

(Brown, 1978). Polyphenoloxidase (PPO) isozyme was determined according to the

method (Baaziz et al., 1994).

D- Statistical analyses:

Experimental data were subjected to one-way analysis of variance (ANOVA) and the

differences between means were separated using Duncans multiple rang test and the (L.S.D)

at 5% level of probability using M-state software (Snedecor and Cochran, 1982).

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Results:

1. Identification of causal pathogen: Fungus isolate was obtained from infected tomato leaves and stem showing wilt

symptoms and identified as F. oxysporum, a based on the morphological characteristics.

Table 1: morphological characters of F. oxysporum:

Culture examination

Colonies on MEA after 7 days at 28°C 65–70 mm diameter, often covering the whole Petri dish, of floccose white to pale greyish magenta mycelium, reverse greyish magenta to dark purple, often paler at the margins.

Microscopic examination

Macro conidia Slightly curved (5-16µm in diameter), usually with three septa, occasionally

Micro conidia Abundant, fusiform to kidney-shaped, produced in false heads from short, stout monophialides.

Chlamydoconidia Produced singly or in pairs

Figure (1)

A. Colony of Fusarium oxysporium on MEA. B. Reverse colony of Fusarium oxysporium on MEA.C. light microscope showing stained conidia of Fusarium oxysporium (Mag. power 20×40x). D. light microscope showing conidiophore of Fusarium oxysporium (Mag. power 20×40x). E. light microscope showing conidia of Fusarium oxysporium (Mag. power 20×10x). F. microscope showing conidia of Fusarium oxysporium (Mag. power 20×40x)

E F

D C

B A

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2. Effect of biotic and abiotic agents on disease index:

Also data in table (2) showed that application of S. marcescens was the best inducer which gave highly protection percent (96.29%), followed by G. lucidum (92.19%) and N. muscorum (88.88%). Also the application of A. oryzae, B. subtilis, Di-potassium hydrogen phosphate, Salix extract, salicylic acid, and T. harzianum gave the same protection percent (85.18%), finally Artemisia (55.55%).

Table 2: Effect of tested biotic and abiotic agents on disease index of tomato plants infected with F. oxysporum:

Key of table: B: Bacillus, S: Serratia, N: Nostoc, A: Anabaena, Di-P.H.P.: DI-potassium hydrogen

phosphate, T: Trichoderma, and G. l. f. B.: Ganoderma leucidum fruiting bodies.

Treatment Classes DI

(disease

index)

(%)

Protection (%)

0 1 2 3 4

Control healthy 6 2 0 0 0 6.25 -

Control Infected 0 0 1 3 4 84.37 0

Infected + B. subtilis 6 0 2 0 0 12.5 85.18

Infected + S. marcescens 7 1 0 0 0 3.125 96.29

Infected + N. muscorum 5 3 0 0 0 9.375 88.88

Infected + A. oryzae 4 4 0 0 0 12.5 85.18

Infected + Artemisia 2 4 0 0 2 37.5 55.55

Infected + Salix 4 4 0 0 0 12.5 85.18

Infected + Salicylic acid 4 4 0 0 0 12.5 85.18

Infected + Di-P.H.P. 4 4 0 0 0 12.5 85.18

Infected + T. harzianum 4 4 0 0 0 12.5 85.18

Infected + G. l. f. B. 6 2 0 0 0 6.25 92.19

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Figure (2): classes of wilt disease symptoms caused by Fusarium oxysporum on tomato plant

3- Biochemical indicators of resistance in tomato: 3.1. Total soluble carbohydrates: Data listed in table (3) revealed that, total soluble

carbohydrate contents of shoot and roots in tomato plants were significantly

decreased due to fusarium infection during two stages of growth. It was found that

application of all biotic and abiotic agents caused highly significant increase in total

soluble carbohydrate in shoot and root during both stages compared to infected

control.

0 1 2

0:(no symptoms), 1:(slight yellow of

lower leaves), 2:(moderate yellow

plant), 3:(wilted plant) and

4:(plants severely stunted and

destroyed) 3 4

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Table 3: Effect of tested biotic and abiotic agents on carbohydrate content of infected tomato plants:

Treatment Total soluble carbohydrate content mg/g d. wt. (g)

Shoot Root

Stage1 Stage2 Stage1 Stage2

Control healthy 25.37 65.65 15.89 21.00

Control Infected 10.96 21.45 8.85 8.06

Infected + B. subtilis 26.81 69.66 11.68 30.92

Infected + S. marcescens 23.55 48.89 13.00 17.51

Infected + N. muscorum 21.57 52.32 12.47 23.83

Infected t+ A. oryzae 23.98 68.56 13.56 17.06

Infected + Artemisia 26.67 52.66 16.19 20.02

Infected + Salix 26.23 72.16 13.55 17.93

Infected + salicylic acid 38.55 70.96 19.63 23.07

Infected + Di-P.H.P. 33.92 56.33 22.78 28.92

Infected + T. harzianum 46.92 63.54 29.82 38.24

Infected + G. l. f. B. 65.24 93.75 56.47 64.03

LSD 0.05 10.30 6.35 5.31 6.17


phosphate, T: Trichoderma, and G. l. f. B.: Ganoderma leucidum fruiting bodies. d. wt: dry weight.

3.2. Total soluble protein contents: The results in Table (4) showed that, total

soluble protein contents of shoot and roots in tomato plants were significantly

decreased due to fusarium infection during two stages of growth. It was found that

application of all biotic and abiotic agents caused highly significant increase in total

soluble protein in shoot and root during both stages compared to infected control.

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Table 4: Effect of tested biotic and abiotic agents on Total soluble protein content of tomato plants infected with F. oxysporum:

Treatment Total soluble protein content

mg/g d. wt. (g)

Shoot Root


Control healthy 21.71 26.80 12.59 19.28





Infected t+ A. oryzae 21.81 29.77 15.92 20.08


Infected + Salix 17.69 21.27 12.80 18.28


Infected + Di-P.H.P. 21.44 26.85 17.86 20.25


Infected + G. l. f. B. 22.02 24.12 12.85 16.68

LSD 0.05 1.69 1.85 1.97 3.52


phosphate, T: Trichoderma, and G. l. f. B.: Ganoderma leucidum fruiting bodies. d. wt: dry weight.

3.3. Total phenols: The results in Table (5) indicate that, all tested inducers

significantly increased total phenols compared with control. The highest increase in

shoots was recorded by G. lucidum then (T. harzianum, Salix, Artemisia, Di-

potassium hydrogen phosphate and N. muscorum) respectively. Then S. marcescens,

B. subtilis and finally salicylic acid. Also all treatments, increased total phenols of

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infected tomato root. The highest increase was induced by S. marcescens and B.

subtilis respectively followed by (A. oryzae, salicylic acid, Di-potassium hydrogen

phosphate and T. harzianum) followed by (G. lucidum, Artemisia and finally Salix. Table 5: Effect of tested biotic and abiotic agents on Phenolic compounds of infected plant:

Key of table: B: Bacillus, S: Serratia, N: Nostoc, A: Anabaena, Di-P.H.P.: DI-potassium hydrogen phosphate, T: Trichoderma, and G. l. f. B.: Ganoderma leucidum fruiting bodies. d. wt: dry weight

3.4. Antioxidant enzymes activity:

Results of the present work (table 6) indicated that, tomato plants infected with F.

oxysporum recorded insignificant increases in PPO activity in shoots compared to healthy

tomato plants at the both stages of growth. All applied inducers caused significantly

increased PPO activity compared with infected control throughout the first stage of growth

except Salix extract and Artemisia recorded insignificantly increasing. While at the second

stage all inducers have insignificant increase except B. subtilis was significantly increased.

Treatment Phenolic compounds mg/100g d.wt(g)

Shoot Root


Control healthy 1.019 1.976 1.145 0.286





Infected + A. oryzae 2.924 2.604 3.166 1.220


Infected + Salix 3.524 4.005 1.503 0.800


Infected + Di-P.H.P. 3.362 3.472 2.533 0.994


Infected + G. l. f. B. 4.009 4.475 2.148 0.802

LSD 0.05 0.0815 0.0868 0.1272 0.4699

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For Peroxidase (POD) activity it was found that all tested agent stimulated POD activity of

shoots. Applied of T. harzianum, A. oryzae, Di-potassium hydrogen phosphate, salicylic acid,

G. lucidum, Artemisia and S. marcescens were significant increase POD activity, followed by N.

muscorum, Salix extract and B. subtilis which recorded insignificant increasing compared with

infected control during the experiment. Also results in (table 6) revealed that, tomato plants

infected with Fusarium gave highly significant increases in Superoxide dismutase SOD

activity related to healthy tomato plant during second stage of growth but insignificant

during the first. It was found that all inducers showed, significant increasing in SOD activity

throughout the second stage of growth as the order A. oryzae, G. lucidum, T. Harzianum, S.

marcescens, Salix extract, Di-potassium hydrogen phosphate, Artemisia, N. muscorum, salicylic acid

and finally B. subtilis. But at the first stage insignificant increasing was recorded and the

highest was A. oryzae followed by G. lucidum, S. marcescens, T. Harzianum, salix extract,

Di-potassium hydrogen phosphate, salicylic acid, Artemisia, N. muscorum and finally B.

subtilis.

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Table 6: Effect of tested biotic and abiotic agents on antioxidant enzymes activity of infected plant:

Key of table: B: Bacillus, S: Serratia, N: Nostoc, A: Anabaena, Di-P.H.P.: DI-potassium hydrogen phosphate, T: Trichoderma, and G. l. f. B.: Ganoderma leucidum fruiting bodies. f.wt.: fresh weight. Endogenous hormones:

Results shown in (chart 1) and revealed that contents of GA3 and SA were

markedly decreased in fusarium infected plants than that of healthy ones. At the same

time, a marked increase in the contents of ABA and IAA was observed in infected plants

as being compared with healthy ones. Concerning the effect tested elicitors on the

challenged plants with F. oxysporum; it was found that contents of GA were generally

decreased due to the application of different elicitors except S. marcescens, N. muscorum,

Treatment Polyphenoloxidase (PPO) ug/g. f.wt of shoot

Peroxidase (POD) ug/g. f.wt of shoot

Superoxide dismutase (SOD) ug/g. f.wt of

shoot

Stage1 Stage2 Stage1 Stage2 Stage1 Stage2

Control healthy 0.14 0. 2 0.31 0.17 0.07 0.08

Control Infected 0.20 0. 5 0.39 0.43 0.37 0.86

Infected + B. subtilis 1.38 3.1 0.51 1. 10 0.50 1.50

Infected + S. marcescens 0.69 0. 81 0.57 1.61 0.87 2.41

Infected + N. muscorum 0.73 1.3 0.55 1.18 0.52 1.86

Infected + A. oryzae 0.42 0.8 1.07 1.90 0.96 2.99

Infected + Artemisia 0.24 0. 7 0.61 1.20 0.60 1.89

Infected + Salix 0.28 0.6 0.54 1.02 0.81 2.36

Infected + salicylic acid 0.36 1. 2 0.59 1.41 0.65 1.68

Infected + Di-P.H.P. 0.53 0. 7 0.98 1.20 0.74 2.27

Infected + T. harzianum 0.40 1. 1 1.10 1.92 0.85 2.48

Infected + G. l. f. B. 0.76 1. 8 0.92 1.74 0.91 2.53

LSD 0.05 0.128 1. 34 0.18 0.768 0.767 0.450

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T. harzianum and G. lucidum fruiting bodies. Contents of SA were decreased due to using of all

elicitors which the same result of ABA except S. marcescens. Also application of S.

marcescens, N. muscorum, Di-potassium hydrogen phosphate and T. harzianum recorded

increasing in IAA.

Chart 1: Effect of tested biotic and abiotic agents on antioxidant enzymes activity of infected plant:

4.1. Detection of the elicited antifungal protein as response to induction SR: Data listed in Table (7 and 8) and Fig (3) showed that tomato plants treated with tested

inducers and infected with F. oxysporum showed variation in number, molecular weight of

protein bands. The variability analysis among tested inducers appeared 149 protein bands.

1: Control healthy,2: Control Infected,3: Infected + B. subtilis,4: Infected + S. marcescens,5; Infected + N. muscorum,6:nfected + A. oryzae,7: Infected + Artemisia,8: Infected + Salix,9: Infected + salicylic acid,10: infected + Di-potassium hydrogen phosphate,11: Infected + T. harzianum,12: Infected + G. lucidum fruiting bodies. Unit is (mg/100g fresh weight).

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The most prominent specific polypeptide alteration (polymorphic bands) ranged molecular

weight from 272.262 to 44.882 KD with percentage 18.3 %. These bands may be related to

tested inducers. The unique bands ranged molecular weight from 44.269to 39.836KDa with

percentage 1.34%. The prominent polypeptide bands in all inducers (monomorphic or

common polypeptide) were ranged molecular weight from 51.504, to 4.189. kDa with

percentage 80.53 %. These bands may be related to tomato plant.

Table (7): Protein fractions in leaf of fusarium infected tomato plants treated with biotic and abiotic agents using SDS-PAGE

MW 1 2 3 4 5 6 7 8 9 10 11 12 Frequency Polymorphism

272.262 - - + + - + + - - + + + 0.583 Polymorphic

96.111 - - - - - + + + + + + + 0.583 Polymorphic

79.633 - - - - - + + + + + + + 0.583 Polymorphic

51.504 + + + + + + + + + + + + 1.000 Monomorphic

44.882 - - + - - - + - + + + + 0.500 Polymorphic

44.269 - + - - - - - - - - - - 0.083 Unique

39.836 - - - - - + - - - - - - 0.083 Unique

31.527 + + + + + + + + + + + + 1.000 Monomorphic

22.973 + + + + + + + + + + + + 1.000 Monomorphic

15.990 + + + + + + + + + + + + 1.000 Monomorphic

13.494 + + + + + + + + + + + + 1.000 Monomorphic

11.652 + + + + + + + + + + + + 1.000 Monomorphic

8.110 + + + + + + + + + + + + 1.000 Monomorphic

6.273 + + + + + + + + + + + + 1.000 Monomorphic

4.874 + + + + + + + + + + + + 1.000 Monomorphic

4.189 + + + + + + + + + + + + 1.000 Monomorphic

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Table (8): polymorphism of Protein fractions in leaf of fusarium infected tomato plants treated with biotic and abiotic agents using SDS-PAGE

(Figure 3): Protein fractions in leaf of fusarium infected tomato plants treated with biotic and abiotic agents using SDS-PAGE

4.2. Biochemical iso-zymes markers:

4.2.1. Polyphenol oxidase isozyme: Tested inducers and F. oxysporum -infection

treatments appeared variation in number, relative mobility and density polypeptide bands

compared with healthy ones. T. harzianum, Di-potassium hydrogen phosphate and Salix

extract were the more effective followed by S. marcescens, and salicylic acid which gave (4)

Isozymes with strong density bands. While B. subtilis, A. oryzae, and Artemisia gave the

Bands 1 2 3 4 5 6 7 8 9 10 11 12 Polymorphism

%

Mono 10 10 10 10 10 10 10 10 10 10 10 10 80.53

Poly 0 0 2 1 0 3 4 2 3 4 4 4 18.13

Unique 0 1 0 0 0 1 0 0 0 0 0 0 1.34 Total bands 10 11 12 11 10 14 14 12 13 14 14 14 100

Key: 1: Control healthy,2: Control Infected,3: Infected + B. subtilis,4: Infected + S. marcescens,5; Infected + N.

muscorum,6:nfected + A. oryzae,7: Infected + Artemisia,8: Infected + Salix,9: Infected + salicylic acid,10: infected + Di-

potassium hydrogen phosphate,11: Infected + T. harzianum,12: Infected + G. lucidum fruiting bodies.

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same number of bands (3isozyme). G. lucidum was the least effective which gave 2 bands

(Table9 and fig4). Table (9): Polyphenol oxidase isozyme of fusarium infected tomato plants treated with biotic and abiotic

(Figure4): Polyphenol oxidase isozyme of infected tomato plants treated with elicetors

4.2.2. Peroxidase isozyme: Applied inducers and F. oxysporum -infection treatments

showed variation in number, relative mobility and density polypeptide bands compared with

healthy ones. A. oryzae was the best inducers which gave 7 Isozymes with strong density

bands. while B. subtilis, N. muscorum , Artemisia , Salix extract and T. harzianum gave the

same number of bands (6 Isozyme) with strong density. S. marcescens, salicylic acid, Di-

potassium hydrogen phosphate and G. lucidum were the least effective which gave the

lowest number of bands (5 Isozyme) with moderate density.

Poly Phenyl

Oxidase Groups

Relative Mobility (R.M) 1 2 3 4 5 6 7 8 9 10 11 12

PX1 0.417 + + - + - - - + + + + - PX2 0.589 + + + + + + + + + + + + PX3 0.863 + + + + + + + + + + + - PX4 0.811 + + + + + + + + + + + +

Key: 1: Control healthy,2: Control Infected,3: Infected + B. subtilis,4: Infected + S. marcescens,5; Infected + N. muscorum,6:nfected + A. oryzae,7: Infected + Artemisia,8: Infected + Salix,9: Infected + salicylic acid,10: infected + Di-potassium hydrogen phosphate,11: Infected + T. harzianum,12: Infected + G. lucidum fruiting bodies.

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Table (10): Peroxidase isozyme of fusarium infected tomato plants treated with biotic and abiotic

(Figure5): Polyphenol oxidase isozyme of fusarium infected tomato plants treated with

biotic and abiotic:

Discussion: Induced resistance can be activated by microorganisms as rhizobacteria in some plants,

while in other plants; the same kind of defense can be induced by certain groups of chemicals

(Prasad and Naik, 2003). The objectives of this study were induction of systemic resistance

in Tomato plants against wilt disease caused by F. oxysporum infection. The first standard to

govern the occurrence of systemic resistance in tomato plants, reduction disease index. Data

Peroxidase Groups

Relative Mobility (R.M) 1 2 3 4 5 6 7 8 9 10 11 12

PX1 0.188 - + + + + + - + + - + - PX2 0.360 + + + + + + + + + + + + PX3 0.531 + + + + + + + + + + + + PX4 0.760 + + + + + + + + + + + + PX5 0.877 + + + + + + + + + + + + PX6 0.943 + + + - + + + - - - - - PX7 0.970 - - - - - + + + - + + -

Key: 1: Control healthy,2: Control Infected,3: Infected + B. subtilis,4: Infected + S. marcescens,5; Infected + N. muscorum,6:nfected + A. oryzae,7: Infected + Artemisia,8: Infected + Salix,9: Infected + salicylic acid,10: infected + Di-potassium hydrogen phosphate,11: Infected + T. harzianum,12: Infected + G. lucidum fruiting bodies.

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obtained in the present study showed that application of all inducers reduced disease index

present which recorded highly protection percent effect against fusarium wilt disease. These

results agree with Chandra et al, 2007 and Farrag et al, 2017 how showed Application of S.

marcescens, B. subtilis ,T. harzianum, N. muscorum as well as, G. lucidum showed highly

significant reduction in percent disease infection compared with control plants infected with

fusarium oxysporum. This result may be explained by Vejan et al., 2016 which reported that

PGPR show synergistic and antagonistic interactions with microorganisms within the

rhizosphere which indirectly boosts plant growth rate or through production of phytohormones

(Bhardwaj D. et al., 2014) or through salicylic acid (SA) accumulation (Van Loon et al.,

1998) which linked with pathogenesis-related (PR) protein accumulation, mainly PR-1 (Dong,

1998). New induced proteins were found in plants treated with tested inducers have been

defined as pathogenesis related proteins, they implicated in plant defense because of their anti-

pathogenic activities (Van-Loon et al., 1994).

Also these results could be demonstrated by Shi et al., 2013 who decided that G.

lucidum fruiting bodies have bioactive compounds Investigations on the mechanisms of

disease suppression. Plant extracts have suggested that the active principles may either act on

the pathogen directly (Amadioha 2000) or induce systemic resistance in host plants resulting

in a reduction of the disease development (Kagale et al. 2004). These results indicated that

salicylic acid showed highly significant reduction in percent disease which explained by Vlot

et al., 2009 who proved that SA play important roles in lignin biosynthesis and regulate plant

responses to pathogen attacks. Our results showed that Di-potassium hydrogen phosphate

highly significant reduction in percent disease which can be demonstrated by two

mechanisms: the first is a direct toxic action on the pathogen and the second in indirect action

due to phosphate anion activates plant defense responses (Moor et al., 2009).

Tested biochemical parameters were highly affected by application of tested elicitors

as total soluble carbohydrate, protein and phenols could be act as indicators of resistance

(Couee et al., 2006, Sudhakar et al., 2007, Rakib and Mustafa 2013, Osman et al., 2016

and Attia et al 2017). However SOD, PO and PPO activities were greater in the

infected plants treated with tested inducers compared to control plants which agree with

(Harish et al., 2009 and Attia, et al 2017). On the other hand this study indicated that the

activities of tested isozymes in challenged plants treated with inducers were higher than that in

infected control which might be the potential factor for induction of SAR against F.

oxysporum according to previous findings. Also, biotic inducers increased many PR-proteins

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such as isozymes of peroxidase and Polyphenol oxidase, (Anand et al., 2009 and Sharaf et al

2016). Finally the present study indicated that all tested biological, Natural and chemical

inducers Induce resistance in tomato plant against wilt disease caused by F. oxysporum.

References: Adebolu ,T. T. and Oladime, j. i. S. A., (2007 ): Antimicrobial activity of leaf extracts of

Ocimumgratissimumon selected diarrheacausing bacteria in southwestern Nigria.African Journal of Biotechnology vol. 6,1; PP. 13-14.

Ajigbola, C. F. and Babalola, O. O., (2013): Integrated Management Strategies for Tomato Fusarium Wilt. Biocontrol Sciences. Vol. 18 (3): pp 117-127.

Akram W., Mahboob A. and Javel A. A., (2013): Bacillus thuringiensis strain 199 can induce systemic resistance in tomato against Fusarium wilt .Europ.J.of Mirobiol. And Immunol., 275-280.

Amadioha, A. C., (2003): Evaluation of some extract against Colletotrichum lindemuthianum on cowpea. Acta Phytopathologica et Entomologica Hungarica, 38:259-265.

Amini, J. and Sidovich, D. F., (2010): The effects of fungicides on Fusarium oxysporum f. sp. lycopersici associated with Fusarium wilt of tomato. Journal of Plant Protection Research 50 (2), 175.

Anand R., Kulothungan S., Karthika S., Sentila R. and Bhuvaneswari K., (2009): Assay of chitinase and beta -1,3 glucanase in Gossypium hirsutum seedlings by Trichoderma spp. against Fusarium oxysporum. International J. Plant Sci. 4: 255-258.

Attia M. S., Abd El-Monem M. A. S. and Ahmed S. Z., (2017): Protective action of some bio-pesticides against early blight disease caused by Alternaria Solani in tomato plant. JISET International Journal of Innovative Science, Engineering and Tech. 4 67-94 ISSN (Online) 2348 – 7968.

Baaziz M., Aissam F., Brakez Z., Bendiab K., El- Hadrami Cheikh R., (1994): Electrophoretic patterns of acid soluble proteins and active isoforms of peroxidase and polyphenol oxidase typifying calli and somatic embryos of two reputed date palm cultivars in Morocco. Euphytica, 76: 159-168.

Bhardwaj D., Ansari M. W., Sahoo R. K. and Tuteja N., (2014): Biofertilizers function as key player in sustainable agriculture by improving soil fertility, plant tolerance and crop productivity.Microb Cell Fact 13: 66.

Brown A. H. D., (1978): Isozymes, plant population, genetic structure and genetic conservation. Theoretical and Applied Genetic, 52:145-157.

Chandra A., Saxena R., Dubey A., Saxena P., (2007): Changes in phenylalanine ammonia lyase activity and isozyme patterns of polyphenol oxidase and peroxidase by salicylic acid leading to enhanced resistance in cowpea against Rhizoctonia solani. Acta Physiologiae Plantarum 29:361-367.

66


https://www.ncbi.nlm.nih.gov/pubmed/24885352





www.ijiset.com

Couee I., Sulmon, C., Gouesbet G. and El Amrani A., (2006): In volvement of soluble sugars in reactive oxygen species balance and responses to oxidative stress in plants. J. Exp. Bot. 57, 449–459. doi: 10.1093/jxb/erj027.

Daniel H. D. and George, C. M., (1972): Peach seed dormancy in relation to endogenous inhibitors and applied growth substances. J. Amer. Soc. Hort. Sci. 97:651-654.

Dong X., (1998): SA, JA, ethylene and disease resistance in plants. Curr, Opin. Plant Biol. 1,316-323.

Farag A. A., Mohamed. S. Attia, Ahmed Younis and Amer M. Abd Elaziz, (2017): Potential impacts of elicitors to improve tomato plant disease resistance. Al Azhar Bulletin of Science Vol,9th., Conf., March 2017, P. 311-321.

Goussous S.J., Abu-El-Samen F. M. and Tahhan R. A., (2010): Antifungal activity of several

medicinal plants extracts against the early blight pathogen (Alternaria solani). Archives of

Phytopathology and Plant Protection, 43: 1746–1758.

Harish S., Kavino M., Kumar N., Balasubramanian P. and Samiyappan R., (2009): Induction of defense-related proteins by mixtures of plant growth promoting endophytic bacteri against Banana bunchy top virus. Biological Control. 51:16–25.

Hibar, K., Edel-Herman, V., Steinberg, C., Gautheron, N., Daami-Remadi, M.,

Alabouvette, C., and El Mahjoub, M., (2007): Genetic Diversity of Fusarium oxysporum

Populations Isolated from Tomato Plants in Tunisia. Journal of Phytopathology 155: 136-142.

Kagale S., Marimuthu T., Thayumanavan B., Nandakumar R., and Samiyappan R.,

(2004): Antimicrobial activ activity and induction of systemic resistance in rice by leaf extract of

Datura metel against Rhizoctonia solani and Xanthomonas oryzae pv. oryzae. Physiological and

Molecular Plant Pathology, 65: 91–100.

Katan, T., Zamir, D., Sarfati, M., and Katan, J., (1991): Vegetative compatibility groups and subgroups in Fusarium oxysporum f. sp. radicislycopersici. Phytopathology 81: 255-262.

Knegt E. and Brunima J., (1973): Rapid sensitive and accurate determination of indole-3acetic acid. Phytochem.,12:573-576.

Leath, R. T, Lukezic, I. and Levine R. G., (1989): Interaction of Fusarium avenaceum and Pseudomonas virdiflava in root rot red clover phytopathology. 79:436-440.

Leslie, J. F., and Summerell, B. A., (2006): The fusarium laboratory manual. UK: Blackwell Publishing Ltd.

Lowery, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., (1951): Protein

measurement with the folin reagent. J. Biol.Chem.193:265-275.

Marklund, S. and Marklund, G., (1974): Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem . 47, 469-474.

67




www.ijiset.com

Matta, A. and Dimond A. E., (1963): Symptoms of Fusarium within relation to quantity of fungus and

enzyme activity in tomato stems .Phytopathol., 53 : 544 – 578.

Moor U., Poldma P., Tonutare T., Karp K., Starast M. and Vool E., (2009): Effect of phosphite fertilization on growth, yield and fruit composition of strawberries. Sci. Hortic., 119(2): 264-269.

Mukherjee S. P. and Choudhuri, M. A., (1983): Implication of water stress-induced changes in the level of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings Pjysiol. Plant. 58:166-170.

Nelson, P. E., Toussoun, T. A., and Marasas, W. F. O., (1983): Fusarium species. An Illustrated Manual for Identification,” The Pennsylvania State University Press, USA, University Park and London, UK, 193 pp.

Olivieri, F. P., Feldman, M. L., Machinandiarena, M. F., Lobato, M. C., Caldiz, D. O., Daleo, G. R. and Andreu, A. B., (2012): Phosphite Applications Induce Molecular Modifications in Potato Tuber Periderm and Cortex That Enhance Resistance to Pathogens. Crop Protection, 32, 1-6.

Osman M. M., Khalifa A. S., Yousri Mutasim A. E., Massaad S. O., Gasemelseed M. M., (2016): In Silico Analysis of Single Nucleotide Polymorphisms (Snps) in Human FTO Gene. J Bioinform, Genomics, Proteomics 1(1): 1003.

Pieterse, C. M. J. and Van Loon, L. C., (2007): Signaling cascades involved in induced resistance. Pp. 65-88 in D. Walters, A. Newton, and G. Lyon, eds. Induced resistance for plant defense: A sustainable approach to crop protection. UK: Blackwell Publishing.

Prasad Y. and Naik M. K., (2003): Evaluation of genotypes, fungicides and plant extracts against early blight of tomato caused by Alternaria solani, Indian Journal of Plant Protection, 31: 49 – 53.

Ragaa, A. Hamouda and Mostafa S. M. El-Ansary, (2013): Biocontrol of Root knot Nematode, Meloidogyne incognita infected banana plants by Cyanobacteria Egypt. J. Agronematol., Vol. 12, No.1, PP. 113-129.

Rakib A., Mustafa A. Athab, and Oadi N. Matny, (2013): Management of potato virus Y (PVY) in potato by some biocontrol agents under field conditions. Journal of Agricultural Technology 9(4):855-861.

Said, A., Naguib, M. I. and Ramzy, M. A., (1964): Sucrose determination as a mean of estimations of the "Drow Back Tax" on exported Halawa Tehinia., Bull. fac. sci., Cairo Univ., 39: 209.

Sharaf A. M. A, Kailla A. M., Attia M. S. and Nofal M. M., (2016): Evaluation of biotic and abiotic elicitors to control Meloidogyne incognita infecting tomato plants. Nat Sci;14 ISSN 1545-0740 (print); ISSN 2375-7167.

Shi M., Zhang Z. and Yang Y., (2013): Antioxidant and immunoregulatory activity of Ganoderma lucidum polysaccharide. Carbohydr. Polym. 95, 200-206.

68




www.ijiset.com

Sivaprakasam E., Balakumar R. and Kavitha D., (2011): Evaluation of antibacterial and antifungal activity of Ganoderma lucidum (Curtis) P. Karst fruit bodies extracts. World J Sci Tech 1: 8–11.

Snedecor, G.W. and Cochran W.G., (1982): Statistical. Methods 6th Edn. Iowa State University Press, Ames. Iowa.

Srivastava, S. K., (1987): Peroxidase and polyphenoloxidase in Brassica junceaplants infected with Macrophominaphaseolina (Tassi. Goid.) and their implication in disease resistance. Phytopathol., 120: 249-254.

Studier, F.W., (1973): Analysis of bacteriophage T7 early RNA and protein of slab gels. Molecular Biol. 79:237-248.

Sudhakar N., Nagendra-Prasad D., Mohan N. and Murugesan K., (2007): Induction of systemic resistance in Lycopersicon esculentum cv. PKM1 (tomato) against Cucumber mosaic virus by using ozone. J Virol Meth 139: 71–77.

Summeral B. A., Salleh B. and Leslie J. F., (2003): A utilitarian approach to Fusarium identification. Plant Dis 87:117–128.

Tassara C., Zaccaro M. C., Storni M. M., Palma M., and Zulpa G., (2008): Biological control of lettuce white mold with cyanobacteria. Int. J.Agri. Biol. 10:487-492.

Umbriet, W. W., Burris, R. H., Stauffer, J. F., Cohen, P. P., Johsen, W. J., Lee page, G. A. Patter, V. R. and Schneicter, W. C., (1969): Manometric techniques, manual describing methods applicable to the studs of tissue metabolism. Burgess publishing co.,

U.S.A; P.P.239.

Van Loon L. C., Pierpoint W. S., Boller T. and Conejero V., (1994): Recommendations for naming plant pathogenesis related proteins. Plant Molecular Biology Reporter. 12: 245-264.

Van Loon L.C., Bakker P. A. H. M. and Pieterse C. M. J., (1998): Systemic resistance induced by rhizosphere bacteria. Annu. Rev. Phytopamol., 36: 453-483.

Vejan P., Abdullah R., Khadiran T., Ismail S. and Boyce A. N., (2016): Role of Plant Growth Promoting Rhizobacteria in Agricultural Sustainability-A Review.Molecules, 21: 573 ), pp. 1-17

Vlot A. C., Depsey D. A. and Klessig D. F., (2009): Salicylic acid, a multifaceted hormone to combat disease. Annual Review of Phytopathology, 47, 177-206.

Younis A., Jennifer S., Fang-Sheng Wu, Hussien El Shikh, Fathy H., Mahmoud E., (2014): Effectiveness of different solvents extracts from edible mushrooms in inhibiting the growth of tumor cells. Cancer Biology Journal, 4(4) P. 1-15. http://www.cancerbio.net/cb/cb0404/001_27172cb040414_1_15.

69


http://www.cancerbio.net/cb/cb0404/001_27172cb040414_1_15.

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